CHARACTERIZATION OF MUTATIONS INDUCED BY ETHYLNITROSOUREA IN SEMINIFEROUS TUBULE GERM-CELLS OF TRANSGENIC B6C3F(1) MICE

Transgenic B6C3F(1) mice carrying a lad target gene were exposed to acute and multiple doses of ethylnitrosourea (ENU), and germ cells from the seminiferous tubules were assayed for mutation 3 and 90 days after treatment. Relative to untreated controls, the mutation frequency increased 3.2- and 19.9-fold at 3 and 90 days after treatment, respectively. Mutant lad genes recovered from untreated and treated groups were sequenced, and the spectra of mutations were determined. Eighty-five percent (11/13) of the spontaneous mutations resulted in G.C --> A.T transitions, all of which occurred at CpG dinucleotides. Fifteen of 22 sites (68%) found mutated 3 days after ENU treatment occurred at G.C base pairs, although some of these are expected to be spontaneous mutations. Ninety days after treatment, 13 of 19 sites (68%) found mutated occurred at A.T base pairs. The mutation spectra seen are consistent with proposed mechanisms of ENU mutagenesis and correlate with the in vivo spectra seen in ENU studies by using transmissibility assays and the hprt gene. These findings represent significant progress toward defining the in vivo spectra of ENU mutagenesis in mammalian germ cells.